Contrary to previous expectations, certain types of proteolytic activities are found frequently in antibodies and their L chain subunits. In autoimmune diseases, the activity of autoantigen-specific proteolytic antibodies is increased and that of nonspecific antibodies is decreased. The long term goals are to define the molecular events and selection pressures leading to development of catalytic activity in antibodies, decipher the biological functions of catalytic antibodies, and develop catalytic antibody-based diagnostic and therapeutic strategies. The purpose of this competitive renewal proposal is to: (a) Identify the contribution of germline V genes and somatic sequence diversification processes in the development of proteolytic activity in antibody VL domains; (b) Establish increased synthesis of proteolytic VL domains in autoimmune disease, determine the correlation of this process with germline V gene usage and somatic hypermutation, and define the upper limits of potency and specificity expressed by autoantibodies; and (c) Redirect the proteolytic specificity of the VL domain by linking it at various of VH domains. Proteolytic activities in recombinant VL domain libraries from unimmunized mice and mice hyperimmunized with L- and D-isomers of an autoantigenic polypeptide will be analyzed. Study of the MRL/lpr mouse strain and of a lupus patient will show whether autoimmune disease is associated with a genetic bias toward proteolytic VL domain synthesis. The results of the D-peptide immunization will show whether somatic processes can elaborate the catalytic sites de novo. Comparison of the sequences of selected catalysts with germline V genes will reveal whether unmutated VL domains express primitive proteolytic activity and whether the somatic hypermutation process leads to specialization of activity. Study of germline reversion mutations in a model VL domain will define the role of individual residues in evolution of the activity. Selected VL domains will be hybridized with antigen-binding VH domains libraries. Study of cleavage of the antigen and unrelated polypeptides by the resultant FV constructs will permit evaluation of the effect of the VH domain on the magnitude and specificity of catalysis. The proposed studies will lead to better understanding of the origin and molecular maturation of proteolytic antibodies made in experimental immunization and autoimmune diseases, and suggest new ways to engineer substrate-specific proteolytic antibodies.